Development of a Live-Cell Imaging Assay to Elucidate Spatiotemporal Dynamics of Extracellular Vesicle Fusion with
Jasper van den Ende1, Kyra A Y Defourny1, Huib H Rabouw2
1Division of Infection Biology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Abstract:
Cells communicate via extracellular vesicles (EVs) containing functional RNAs, proteins, and lipids. Knowledge on the fate of internalized EVs, especially their capacity to fuse with target cell membranes and deliver luminal cargo, is limited. Currently available EV-cargo delivery assays are indirect and thus unlikely to uncover molecular players and conditions that specifically control the EV-fusion step. Here, we present a novel live-cell imaging assay for detection of EV-binding, -uptake, and -fusion in time and space. We employed the SunTag system for exceptional signal amplification. EV-donor cells were engineered to tag the luminal EV-membrane with a fluorescent label coupled to SunTag peptides. Recipient cells express fluorescent single-chain anti-SunTag antibody (STAb), which binds EV-enclosed SunTag upon its cytosolic exposure. Using SunTagged EVs carrying fusogen VSV-G, we visualize the EV-fusion process, quantify fusion kinetics and efficiency, and determine subcellular localization of fusion events. We term this methodology the Extracellular Vesicle Fusion Spatiotemporal Imaging Method (EV-FUSIM). In the future, this technology can support the identification of fusogenic EV-subsets, as well as molecular players and drugs that modulate EV-fusion, without confounding effects of post-fusion processes. This will extend knowledge on EV-biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads.
